Method Development and Validation for Simultaneous Detection of Corticosteroids, Small Peptides, SARMs and Quaternary Ammonium Drugs in Camel Urine for Doping Control Applications

Background: Detection and identification of a wide range of drugs, including small peptides, corticosteroids, selective androgen receptor modulators (SARMs), and quaternary ammonium drugs (QADs), are imperative across several domains, particularly in anti-doping analysis, given the potential misuse of these substances in animal sports, there is an urgent demand for an allencompassing screening method to effectively identify these compounds. Objective: To develop a robust and sensitive high-resolution method for simultaneous screening of small peptides, corticosteroids, SARMs, and QADs using liquid chromatography accurate mass spectrometry in post-race urine samples. This method integrates a streamlined single-stage extraction approach, significantly enhancing efficiency and adaptability for screening drugs across various classes. Method: The method development and validation involved a comprehensive solid-phase extraction protocol, which included a sequential elution for corticosteroids, small peptides, SARMs, and QADs. Chromatographic separation was achieved using a reverse-phase C18 column, and the analysis was performed using liquid chromatography - high-resolution accurate mass spectrometry. Results: The developed method was validated using a selection of drugs representing each class. The method exhibited robustness and sensitivity, enabling the simultaneous screening of the specified drug classes. The flexibility inherent in the proposed extraction and analysis method allows for seamless integration of new drug candidates eliminating the need for method redevelopment. Conclusion: A versatile and effective screening method was successfully developed and validated for the simultaneous detection of small peptides, corticosteroids, SARMs, and QADs. The method's ability for retrospective analysis of emerging drugs using full scan HRMS enhances its utility. This method holds great promise across various fields where accurate and comprehensive drug screening is imperative.